Antioxidative long chain alkylresorcinols. Synthesis and deuterium labelling of bioactive compounds present in whole grains

The first synthesis of long chain 5-n-alkylresorcinols (C15-C25) in whole grains and whole grain products by a novel modification of Wittig reaction is described. 5-n-Alkylresorcinols are phenolic lipids that have various effects on biological systems, such as antioxidant activity and interaction with biological membranes. These compounds are considered as biomarkers of whole grain intake, which is connected with reduced risk of cardiovascular diseases and certain cancers. Novel hapten derivatives of 5-n-alkylresorcinols, potential compounds for immunoanalytical techniques, are prepared by the same procedure utilizing microwave catalysed aqueous Wittig reaction as the key step. The synthesised analogues are required by various analytical, metabolism and bioactivity investigations. Four alternative strategies for producing deuterium polylabelled 5-n-alkylresorcinols are explored. Ring-labelled D3-alkylresorcinols were synthesized by acidic H/D exchange. Side chain -labelled D4-derivative was prepared by a total synthesis approach utilizing D2 deuterogenation of a D2-alkene derivative, and deuterogenation of alkynes was investigated in another total synthesis approach. An -D3-labelled alkylresorcinol is isotopically pure and completely stable under all relevant conditions encountered during analytical work. The labelling of another phenolic component of whole grains was explored. The preparation of D3-ferulic acid and related compounds by way of selective methylation of the precursors is described. The deuterated compounds are useful as standards in the quantification of these natural products in various substances, such as food and human fluids. The pure 5-n-alkylresorcinol analogues prepared were used in in vitro experiments on alkylresorcinol antioxidant activity and antigenotoxicity. The in vitro experiments show that alkylresorcinols act as antioxidants, especially when incorporated into biological systems, but possess lower activity in chemical tests (FRAP and DPPH assay). Whole grain alkylresorcinols are shown for the first time to have a protective effect against copper induced oxidation of LDL, and H2O2 or genotoxic faecal water induced damage on HT29 cells.

A quantum chemical investigation of the metal centres in cytochrome c oxidase

Quantum effects are often of key importance for the function of biological systems at molecular level. Cellular respiration, where energy is extracted from the reduction of molecular oxygen to water, is no exception. In this work, the end station of the electron transport chain in mitochondria, cytochrome c oxidase, is investigated using quantum chemical methodology. Cytochrome c oxidase contains two haems, haem a and haem a3. Haem a3, with its copper companion, CuB, is involved in the final reduction of oxygen into water. This binuclear centre receives the necessary electrons from haem a. Haem a, in turn, receives its electrons from a copper ion pair in the vicinity, called CuA. Density functional theory (DFT) has been used to clarify the charge and spin distributions of haem a, as well as changes in these during redox activity. Upon reduction, the added electron is shown to be evenly distributed over the entire haem structure, important for the accommodation of the prosthetic group within the protein. At the same time, the spin distribution of the open-shell oxidised state is more localised to the central iron. The exact spin density distribution has been disputed in the literature, however, different experiments indicating different distributions of the unpaired electron. The apparent contradiction is shown to be due to the false assumption of a unit amount of unpaired electron density; in fact, the oxidised state has about 1.3 unpaired electrons. The validity of the DFT results have been corroborated by wave function based coupled cluster calculations. Point charges, for use in classical force field based simulations, have been parameterised for the four metal centres, using a newly developed methodology. In the procedure, the subsystem for which point charges are to be obtained, is surrounded by an outer region, with the purpose of stabilising the inner region, both electronically and structurally. Finally, the possibility of vibrational promotion of the electron transfer step between haem a and a3 has been investigated. Calculating the full vibrational spectra, at DFT level, of a combined model of the two haems, revealed several normal modes that do shift electron density between the haems. The magnitude of the shift was found to be moderate, at most. The proposed mechanism could have an assisting role in the electron transfer, which still seems to be dominated by electron tunnelling.

Intermolecular Interactions of Noble-Gas-Containing Species

The importance of intermolecular interactions to chemistry, physics, and biology is difficult to overestimate. Without intermolecular forces, condensed phase matter could not form. The simplest way to categorize different types of intermolecular interactions is to describe them using van der Waals and hydrogen bonded (H-bonded) interactions. In the H-bond, the intermolecular interaction appears between a positively charged hydrogen atom and electronegative fragments and it originates from strong electrostatic interactions. H-bonding is important when considering the properties of condensed phase water and in many biological systems including the structure of DNA and proteins. Vibrational spectroscopy is a useful tool for studying complexes and the solvation of molecules. Vibrational frequency shift has been used to characterize complex formation. In an H-bonded system A∙∙∙H-X (A and X are acceptor and donor species, respectively), the vibrational frequency of the H-X stretching vibration usually decreases from its value in free H-X (red-shift). This frequency shift has been used as evidence for H-bond formation and the magnitude of the shift has been used as an indicator of the H-bonding strength. In contrast to this normal behavior are the blue-shifting H-bonds, in which the H-X vibrational frequency increases upon complex formation. In the last decade, there has been active discussion regarding these blue-shifting H-bonds. Noble-gases have been considered inert due to their limited reactivity with other elements. In the early 1930 s, Pauling predicted the stable noble-gas compounds XeF6 and KrF6. It was not until three decades later Neil Bartlett synthesized the first noble-gas compound, XePtF6, in 1962. A renaissance of noble-gas chemistry began in 1995 with the discovery of noble-gas hydride molecules at the University of Helsinki. The first hydrides were HXeCl, HXeBr, HXeI, HKrCl, and HXeH. These molecules have the general formula of HNgY, where H is a hydrogen atom, Ng is a noble-gas atom (Ar, Kr, or Xe), and Y is an electronegative fragment. At present, this class of molecules comprises 23 members including both inorganic and organic compounds. The first and only argon-containing neutral chemical compound HArF was synthesized in 2000 and its properties have since been investigated in a number of studies. A helium-containing chemical compound, HHeF, was predicted computationally, but its lifetime has been predicted to be severely limited by hydrogen tunneling. Helium and neon are the only elements in the periodic table that do not form neutral, ground state molecules. A noble-gas matrix is a useful medium in which to study unstable and reactive species including ions. A solvated proton forms a centrosymmetric NgHNg+ (Ng = Ar, Kr, and Xe) structure in a noble-gas matrix and this is probably the simplest example of a solvated proton. Interestingly, the hypothetical NeHNe+ cation is isoelectronic with the water-solvated proton H5O2+ (Zundel-ion). In addition to the NgHNg+ cations, the isoelectronic YHY- (Y = halogen atom or pseudohalogen fragment) anions have been studied with the matrix-isolation technique. These species have been known to exist in alkali metal salts (YHY)-M+ (M = alkali metal e.g. K or Na) for more than 80 years. Hydrated HF forms the FHF- structure in aqueous solutions, and these ions participate in several important chemical processes. In this thesis, studies of the intermolecular interactions of HNgY molecules and centrosymmetric ions with various species are presented. The HNgY complexes show unusual spectral features, e.g. large blue-shifts of the H-Ng stretching vibration upon complexation. It is suggested that the blue-shift is a normal effect for these molecules, and that originates from the enhanced (HNg)+Y- ion-pair character upon complexation. It is also found that the HNgY molecules are energetically stabilized in the complexed form, and this effect is computationally demonstrated for the HHeF molecule. The NgHNg+ and YHY- ions also show blue-shifts in their asymmetric stretching vibration upon complexation with nitrogen. Additionally, the matrix site structure and hindered rotation (libration) of the HNgY molecules were studied. The librational motion is a much-discussed solid state phenomenon, and the HNgY molecules embedded in noble-gas matrices are good model systems to study this effect. The formation mechanisms of the HNgY molecules and the decay mechanism of NgHNg+ cations are discussed. A new electron tunneling model for the decay of NgHNg+ absorptions in noble-gas matrices is proposed. Studies of the NgHNg+∙∙∙N2 complexes support this electron tunneling mechanism.

Optical Tweezers for Single Molecule Biology

Molecular machinery on the micro-scale, believed to be the fundamental building blocks of life, involve forces of 1-100 pN and movements of nanometers to micrometers. Micromechanical single-molecule experiments seek to understand the physics of nucleic acids, molecular motors, and other biological systems through direct measurement of forces and displacements. Optical tweezers are a popular choice among several complementary techniques for sensitive force-spectroscopy in the field of single molecule biology. The main objective of this thesis was to design and construct an optical tweezers instrument capable of investigating the physics of molecular motors and mechanisms of protein/nucleic-acid interactions on the single-molecule level. A double-trap optical tweezers instrument incorporating acousto-optic trap-steering, two independent detection channels, and a real-time digital controller was built. A numerical simulation and a theoretical study was performed to assess the signal-to-noise ratio in a constant-force molecular motor stepping experiment. Real-time feedback control of optical tweezers was explored in three studies. Position-clamping was implemented and compared to theoretical models using both proportional and predictive control. A force-clamp was implemented and tested with a DNA-tether in presence of the enzyme lambda exonuclease. The results of the study indicate that the presented models describing signal-to-noise ratio in constant-force experiments and feedback control experiments in optical tweezers agree well with experimental data. The effective trap stiffness can be increased by an order of magnitude using the presented position-clamping method. The force-clamp can be used for constant-force experiments, and the results from a proof-of-principle experiment, in which the enzyme lambda exonuclease converts double-stranded DNA to single-stranded DNA, agree with previous research. The main objective of the thesis was thus achieved. The developed instrument and presented results on feedback control serve as a stepping stone for future contributions to the growing field of single molecule biology.

A metabolomic approach to studying mechanisms of polymeric gene delivery to retinal pigment epithelial cells

Tiivistelmä ReferatAbstract Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.

Agroforestry systems for sustainable livelihoods and improved land management in the East Usambara Mountains, Tanzania

Forest destruction for agriculture continues to be a major threat to the rich biological diversity in the East Usambara Mountains in the north-eastern corner of Tanzania. The highest ratio of endemic plant and animal species found on 100 km2 anywhere in the world is depending on the remaining natural forests. Forests are vitally important for the local population in many different ways, and nationally they are an important source of water and hydroelectricity. The soils, of low fertility and mostly acidic Ferrasols, mainly have the nutrients in the topsoil. After clear-cutting, the soils soon become poor when the topsoil is eroded. High-value cardamom is nowadays unsustainably cultivated in the natural forests of the East Usambaras. The general aim was to study the possibilities to develop new profitable and sustainable agroforestry systems for the benefit of the local people that could contribute to relieving the pressure on the remaining natural forests in the East Usambara Mountains. Results from a spice crop agroforestry trial, established in cooperation with a local farmer, showed a clear advantage of intercropping cardamom (Elettaria cardamomum) and black pepper (Piper nigrum) with trees, especially with Grevillea robusta. The nitrogen fixing tree species Gliricidia sepium also improved the nitrogen and organic matter content of the soil over levels found in the natural forest. With improved agroforestry methods for spice production the households generated as much as13 times the net income obtained with traditional forest cultivation practices. There are thus sustainable and profitable ways to cultivate spices as cash crops in well-managed homegardens. However, the farmers need stable markets, access to credit and comprehensive extension services. The soil fertility depletion should be reversed with organic manure application and an enabling policy environment for the smallholder-farming sector. Strong farmers organisations and equal rights to resources and decision-making are needed. Organic spices have an increasing demand, and their export would be profitable for these farmers. What is, however, most needed for a change is a political will of a government that understands the importance of agricultural and forestry development for poverty reduction.

Tropical dryland agroforestry on clay soils: : Analysis of systems based on Acacia senegal in the Blue Nile region, Sudan

Acacia senegal, the gum arabic producing tree, is the most important component in traditional dryland agroforestry systems in the Blue Nile region, Sudan. The aim of the present study was to provide new knowledge on the potential use of A. senegal in dryland agroforestry systems on clay soils, as well as information on tree/crop interaction, and on silvicultural and management tools, with consideration on system productivity, nutrient cycling and sustainability. Moreover, the aim was also to clarify the intra-specific variation in the performance of A. senegal and, specifically, the adaptation of trees of different origin to the clay soils of the Blue Nile region. In agroforestry systems established at the beginning of the study, tree and crop growth, water use, gum and crop yields, nutrient cycling and system performance were investigated for a period of four years (1999 to 2002). Trees were grown at 5 x 5 m and 10 x 10 m spacing alone or in mixture with sorghum or sesame; crops were also grown in sole culture. The symbiotic biological N2 fixation by A. senegal was estimated using the 15N natural abundance (δ15N) procedure in eight provenances collected from different environments and soil types of the gum arabic belt and grown in clay soil in the Blue Nile region. Balanites aegyptiaca (a non-legume) was used as a non-N-fixing reference tree species, so as to allow 15N-based estimates of the proportion of the nitrogen in trees derived from the atmosphere. In the planted acacia trees, measurements were made on shoot growth, water-use efficiency (as assessed by the δ13C method) and (starting from the third year) gum production. Carbon isotope ratios were obtained from the leaves and branch wood samples. The agroforestry system design caused no statistically significant variation in water use, but the variation was highly significant between years, and the highest water use occurred in the years with high rainfall. No statistically significant differences were found in sorghum or sesame yields when intercropping and sole crop systems were compared (yield averages were 1.54 and 1.54 ha-1 for sorghum and 0.36 and 0.42 t ha-1 for sesame in the intercropped and mono-crop plots, respectively). Thus, at an early stage of agroforestry system management, A. senegal had no detrimental effect on crop yield, but the pattern of resource capture by trees and crops may change as the system matures. Intercropping resulted in taller trees and larger basal and crown diameters as compared to the development of sole trees. It also resulted in a higher land equivalent ratio. When gum yields were analysed it was found that a significant positive relationship existed between the second gum picking and the total gum yield. The second gum picking seems to be a decisive factor in gum production and could be used as an indicator for the total gum yield in a particular year. In trees, the concentrations of N and P were higher in leaves and roots, whereas the levels of K were higher in stems, branches and roots. Soil organic matter, N, P and K contents were highest in the upper soil stratum. There was some indication that the P content slightly increased in the topsoil as the agroforestry plantations aged. At a stocking of 400 trees ha-1 (5 x 5 m spacing), A. senegal accumulated in the biomass a total of 18, 1.21, 7.8 and 972 kg ha-1of N, P, K and OC, respectively. Trees contributed ca. 217 and 1500 kg ha-1 of K and OC, respectively, to the top 25-cm of soil over the first four years of intercropping. Acacia provenances of clay plain origin showed considerable variation in seed weight. They also had the lowest average seed weight as compared to the sandy soil (western) provenances. At the experimental site in the clay soil region, the clay provenances were distinctly superior to the sand provenances in all traits studied but especially in basal diameter and crown width, thus reflecting their adaptation to the environment. Values of δ13C, indicating water use efficiency, were higher in the sand soil group as compared to the clay one, both in leaves and in branch wood. This suggests that the sand provenances (with an average value of -28.07 ) displayed conservative water use and high drought tolerance. Of the clay provenances, the local one (Bout) displayed a highly negative (-29.31 ) value, which indicates less conservative water use that resulted in high productivity at this particular clay-soil site. Water use thus appeared to correspond to the environmental conditions prevailing at the original locations for these provenances. Results suggest that A. senegal provenances from the clay part of the gum belt are adapted for a faster growth rate and higher biomass and gum productivity as compared to provenances from sand regions. A strong negative relationship was found between the per-tree gum yield and water use efficiency, as indicated by δ13C. The differences in water use and gum production were greater among provenance groups than within them, suggesting that selection among rather than within provenances would result in distinct genetic gain in gum yield. The relative δ15N values ( ) were higher in B. aegyptiaca than in the N2-fixing acacia provenances. The amount of Ndfa increased significantly with age in all provenances, indicating that A. senegal is a potentially efficient nitrogen fixer and has an important role in t agroforestry development. The total above-ground contribution of fixed N to foliage growth in 4-year-old A. senegal trees was highest in the Rahad sand-soil provenance (46.7 kg N ha-1) and lowest in the Mazmoom clay-soil provenance (28.7 kg N ha-1). This study represents the first use of the δ15N method for estimating the N input by A. senegal in the gum belt of Sudan. Key words: Acacia senegal, agroforestry, clay plain, δ13C, δ15N, gum arabic, nutrient cycling, Ndfa, Sorghum bicolor, Sesamum indicum

Interactions of nandrolone and psychostimulant drugs on central monoaminergic systems

It has been hypothesized that abuse of supra-therapeutic doses of anabolic androgenic steroids (AASs) can lead to dependence and function as a gateway to abuse of other drugs. This is supported by behavioral studies on animal models and psychiatric evaluations of human subjects, although their neurochemical effects remain largely unknown. A large body of evidence suggests that the ability of the drugs to induce a strong elevation of extracellular dopamine (DA) levels in the nucleus accumbens (NAc), especially, plays a crucial role in their reinforcing effects. -- This study had four main aims. The first was to explore the effects of nandrolone decanoate on dopaminergic and serotonergic activities in the brains of rats. The second aim was to assess whether or not nandrolone pre-exposure modulates the acute neurochemical and behavioral effects of psychostimulant drugs in experimental animals. The third was to investigate if the AAS-pre-treatment induced changes in brain reward circuitry are reversible. And the fourth main goal was to evaluate the role of androgen and estrogen receptors in the modulation of the dopaminergic and serotonergic effects of acute injections of stimulant drugs by sub-chronic nandrolone treatment. The results showed that nandrolone decanoate at doses, high enough to induce erythropoiesis, significantly increased the levels of DOPAC and 5-HT in the cerebral cortex. Co-administration of AAS and psychostimulant drugs showed that the increase in extracellular DA and 5-HT concentration evoked by amphetamine, MDMA and cocaine in the NAc was attenuated dose-dependently by pretreatment with nandrolone. Nandrolone pre-exposure also attenuated the ability of stimulants to cause increased stereotyped behavior and locomotor activity. Despite the significant decrease in nandrolone concentration in blood, the attenuation of cocaine’s effects remained unchanged after a fairly long period without nandrolone, suggesting that nandrolone effects could be long lasting. Blockade of androgen receptors with flutamide abolished the attenuating effect of nandrolone pretreatment on amphetamine-induced elevation of extracellular DA concentration. --- In conclusion, the results show that AAS-pretreatment is able to inhibit the reward-related neurochemical and behavioral effects of amphetamine, MDMA and cocaine in experimental animals. Furthermore, it seems that these effects could be long lasting and it appears that the ability of nandrolone to modulate reward-related effects of stimulants is dependent on activation of androgen receptors.

Lipid assisted membrane entry in specific phospholipid targeted protein systems

The purpose of this thesis project is to study changes in the physical state of cell membranes during cell entry, including how these changes are connected to the presence of ceramide. The role of enzymatical manipulation of lipids in bacterial internalization is also studied. A novel technique, where a single giant vesicle is chosen under the microscope and an enzyme coupled-particle attached to the micromanipulator pipette towards the vesicle, is used. Thus, the enzymatic reaction on the membrane of the giant vesicle can be followed in real-time. The first aim of this study is to develop a system where the localized sphingomyelinase membrane interaction could be observed on the surface of the giant vesicle and the effects could be monitored with microscopy. Domain formation, which resembles acid sphingomyelinase (ASMase), causes CD95 clustering in the cell membrane due to ceramide production (Grassmé et al., 2001a; Grassmé et al., 2001b) and the formation of small vesicles inside the manipulated giant vesicle is observed. Sphingomyelinase activation has also been found to be an important factor in the bacterial and viral invasion process in nonphagocytic cells (Grassmé et al., 1997; Jan et al., 2000). Accordingly, sphingomyelinase reactions in the cell membrane might also give insight into bacterial or viral cellular entry events. We found sphingomyelinase activity in Chlamydia pneumonia elementarybodies (EBs). Interestingly, the bacterium enters host cells by endocytosis but the internalization mechanism of Chlamydia is unknown. The hypothesis is that sphingomyelin is needed for host cell entry in the infection of C. pneumonia. The second project focuses on this subject. The goal of the third project is to study a role of phosphatidylserine as a target for a membrane binding protein. Phosphatidylserine is chosen because of its importance in fusion processes. This will be another example for the importance of lipids in cell targeting, internalization, and externalization.

Mass Spectrometry Informatics in Systems Biology

Mass spectrometry (MS) became a standard tool for identifying metabolites in biological tissues, and metabolomics is slowly acknowledged as a legitimate research discipline for characterizing biological conditions. The computational analyses of metabolomics, however, lag behind compared with the rapid advances in analytical aspects for two reasons. First is the lack of standardized data repository for mass spectra: each research institution is flooded with gigabytes of mass-spectral data from its own analytical groups and cannot host a world-class repository for mass spectra. The second reason is the lack of informatics experts that are fully experienced with spectral analyses. The two barriers must be overcome to establish a publicly free data server for MS analysis in metabolomics as does GenBank in genomics and UniProt in proteomics. The workshop brought together bioinformaticians working on mass spectral analyses in Finland and Japan with the goal to establish a consortium to freely exchange and publicize mass spectra of metabolites measured on various platforms computational tools to analyze spectra spectral knowledge that are computationally predicted from standardized data. This book contains the abstracts of the presentations given in the workshop. The programme of the workshop consisted of oral presentations from Japan and Finland, invited lectures from Steffen Neumann (Leibniz Institute of Plant Biochemistry), Matej Oresic (VTT), Merja Penttila (VTT) and Nicola Zamboni (ETH Zurich) as well as free form discussion among the participants. The event was funded by Academy of Finland (grants 139203 and 118653), Japan Society for the Promotion of Science (JSPS Japan-Finland Bilateral Semi- nar Program 2010) and Department of Computer Science University of Helsinki. We would like to thank all the people contributing to the technical pro- gramme and the sponsors for making the workshop possible. Helsinki, October 2010 Masanori Arita, Markus Heinonen and Juho Rousu

Local systems, global impacts : Using life cycle assessment to analyse the potential and constraints of industrial symbioses

Human activities extract and displace different substances and materials from the earth s crust, thus causing various environmental problems, such as climate change, acidification and eutrophication. As problems have become more complicated, more holistic measures that consider the origins and sources of pollutants have been called for. Industrial ecology is a field of science that forms a comprehensive framework for studying the interactions between the modern technological society and the environment. Industrial ecology considers humans and their technologies to be part of the natural environment, not separate from it. Industrial operations form natural systems that must also function as such within the constraints set by the biosphere. Industrial symbiosis (IS) is a central concept of industrial ecology. Industrial symbiosis studies look at the physical flows of materials and energy in local industrial systems. In an ideal IS, waste material and energy are exchanged by the actors of the system, thereby reducing the consumption of virgin material and energy inputs and the generation of waste and emissions. Companies are seen as part of the chains of suppliers and consumers that resemble those of natural ecosystems. The aim of this study was to analyse the environmental performance of an industrial symbiosis based on pulp and paper production, taking into account life cycle impacts as well. Life Cycle Assessment (LCA) is a tool for quantitatively and systematically evaluating the environmental aspects of a product, technology or service throughout its whole life cycle. Moreover, the Natural Step Sustainability Principles formed a conceptual framework for assessing the environmental performance of the case study symbiosis (Paper I). The environmental performance of the case study symbiosis was compared to four counterfactual reference scenarios in which the actors of the symbiosis operated on their own. The research methods used were process-based life cycle assessment (LCA) (Papers II and III) and hybrid LCA, which combines both process and input-output LCA (Paper IV). The results showed that the environmental impacts caused by the extraction and processing of the materials and the energy used by the symbiosis were considerable. If only the direct emissions and resource use of the symbiosis had been considered, less than half of the total environmental impacts of the system would have been taken into account. When the results were compared with the counterfactual reference scenarios, the net environmental impacts of the symbiosis were smaller than those of the reference scenarios. The reduction in environmental impacts was mainly due to changes in the way energy was produced. However, the results are sensitive to the way the reference scenarios are defined. LCA is a useful tool for assessing the overall environmental performance of industrial symbioses. It is recommended that in addition to the direct effects, the upstream impacts should be taken into account as well when assessing the environmental performance of industrial symbioses. Industrial symbiosis should be seen as part of the process of improving the environmental performance of a system. In some cases, it may be more efficient, from an environmental point of view, to focus on supply chain management instead.